Limits...
Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1.

Schneider M, Scholtka B, Gottschalk U, Faiss S, Schatz D, Berghof-Jäger K, Steinberg P - Cancers (Basel) (2010)

Bottom Line: In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form.B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas.Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. scholtka@uni-potsdam.de.

ABSTRACT
In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243-1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

(A) PCR-RFLP analysis of the K-Ras codon 12 mutation. Lane 1: wild-type control; lane 2: mutation control - codon 12 GGT→GAT; lanes 3–12: human tissue samples No. 44, 64, 6, 18, 13, 43, 1, 4, 69, and 29. Lanes 6 (sample No. 18) and 12 (sample No. 29) exhibit aberrant band motilities (*). (B) Sequencing profiles of DNA fragments isolated from polyacrylamide gel. On the left side: wild-type sequence at codon 12. On the right side: shifted band sequence of sample No. 18. Arrows show the transversion of wild-type guanine (on the left side) to thymine (on the right side) at the first base position of codon 12.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3756351&req=5

f2-cancers-03-00091: (A) PCR-RFLP analysis of the K-Ras codon 12 mutation. Lane 1: wild-type control; lane 2: mutation control - codon 12 GGT→GAT; lanes 3–12: human tissue samples No. 44, 64, 6, 18, 13, 43, 1, 4, 69, and 29. Lanes 6 (sample No. 18) and 12 (sample No. 29) exhibit aberrant band motilities (*). (B) Sequencing profiles of DNA fragments isolated from polyacrylamide gel. On the left side: wild-type sequence at codon 12. On the right side: shifted band sequence of sample No. 18. Arrows show the transversion of wild-type guanine (on the left side) to thymine (on the right side) at the first base position of codon 12.

Mentions: K-Ras gene mutations were screened by RFLP analysis according to the method described by Schimanski et al. [40]. Briefly, 2 μL of the whole genome amplification product (100–300 ng genomic DNA) obtained by using the REPLI-g Mini Kit were PCR-amplified with the oligonucleotide primer pair Ras A and Ras B [19], generating a 166 bp amplicon. The mismatch primer Ras A hybridizing with codons 2–11 of the K-Ras gene exon 1 introduces a restriction site for BstXI or XcmI into the amplicon. In case a template is mutated in codon 12 or 13, the restriction site is lost. The subsequent enzymatic restriction of the wild-type K-Ras leads to an enrichment of mutated DNA strands. Gel bands, the size of the mutated control fragments, were cut out and sequenced. Figure 2 illustrates a typical RFLP analysis of the K-Ras gene and the corresponding sequencing profile of the shifted band.


Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1.

Schneider M, Scholtka B, Gottschalk U, Faiss S, Schatz D, Berghof-Jäger K, Steinberg P - Cancers (Basel) (2010)

(A) PCR-RFLP analysis of the K-Ras codon 12 mutation. Lane 1: wild-type control; lane 2: mutation control - codon 12 GGT→GAT; lanes 3–12: human tissue samples No. 44, 64, 6, 18, 13, 43, 1, 4, 69, and 29. Lanes 6 (sample No. 18) and 12 (sample No. 29) exhibit aberrant band motilities (*). (B) Sequencing profiles of DNA fragments isolated from polyacrylamide gel. On the left side: wild-type sequence at codon 12. On the right side: shifted band sequence of sample No. 18. Arrows show the transversion of wild-type guanine (on the left side) to thymine (on the right side) at the first base position of codon 12.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756351&req=5

f2-cancers-03-00091: (A) PCR-RFLP analysis of the K-Ras codon 12 mutation. Lane 1: wild-type control; lane 2: mutation control - codon 12 GGT→GAT; lanes 3–12: human tissue samples No. 44, 64, 6, 18, 13, 43, 1, 4, 69, and 29. Lanes 6 (sample No. 18) and 12 (sample No. 29) exhibit aberrant band motilities (*). (B) Sequencing profiles of DNA fragments isolated from polyacrylamide gel. On the left side: wild-type sequence at codon 12. On the right side: shifted band sequence of sample No. 18. Arrows show the transversion of wild-type guanine (on the left side) to thymine (on the right side) at the first base position of codon 12.
Mentions: K-Ras gene mutations were screened by RFLP analysis according to the method described by Schimanski et al. [40]. Briefly, 2 μL of the whole genome amplification product (100–300 ng genomic DNA) obtained by using the REPLI-g Mini Kit were PCR-amplified with the oligonucleotide primer pair Ras A and Ras B [19], generating a 166 bp amplicon. The mismatch primer Ras A hybridizing with codons 2–11 of the K-Ras gene exon 1 introduces a restriction site for BstXI or XcmI into the amplicon. In case a template is mutated in codon 12 or 13, the restriction site is lost. The subsequent enzymatic restriction of the wild-type K-Ras leads to an enrichment of mutated DNA strands. Gel bands, the size of the mutated control fragments, were cut out and sequenced. Figure 2 illustrates a typical RFLP analysis of the K-Ras gene and the corresponding sequencing profile of the shifted band.

Bottom Line: In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form.B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas.Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. scholtka@uni-potsdam.de.

ABSTRACT
In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243-1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

No MeSH data available.


Related in: MedlinePlus